Nitroxides as Protectors against Oxidative Stress Summary Nitroxides are proving to have broad utility in a number of disease processes and/or conditions that represent excessive oxidative stress. The fact that nitroxides exert activity over such a range of conditions speaks to the importance of free radical reactions in tissue. Likewise, it is becoming apparent that free radicals are important in normal molecular signaling pathways and related gene expression. We have extensively studied the chemical mechanisms of nitroxides as antioxidants and are presently exploring how nitroxides can influence gene expression. Cells treated with non-toxic concentrations of several different nitroxide analogues; including a nitroxide incapable of cellular entry (and protection) all exhibit similar patterns of gene expression. A comparison was made of gene expression profiles for MCF7 cells exposed to nitroxides and various forms of oxidative stress (hydrogen peroxide, superoxide, nitric oxide, and ionizing radiation). A small subset of genes related to inflammatory responses was up regulated for all forms of oxidative stress; whereas, the same genes for nitroxide treatment were all down regulated, implying that nitroxides may be very effective in reducing inflammation. In collaborative studies, the effects of chronic administration of Tempol (supplemented in food) of two mouse models that exhibit neurodegeneration and/or neurological damage have been evaluated. Iron regulatory protein 2 knockout mice (IRP2-/-) exhibit age-related neurodegeneration (similar to Parkinsons disease patients). Tempol treatment attenuated the progression of neurodegeneration in IRP2-/- mice. Tempol was also shown to be highly protective in an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE mouse model is an acute or chronic demyelinating autoimmune disease whose clinical manifestations of paralysis and quadriparesis that closely resemble those observed in Multiple Sclerosis patients. These preliminary data are exciting and may represent a new approach toward treating these diseases. Lastly, we continue to search for the mechanism(s) of how long-term administration of Tempol (in the food or drinking water) results in dramatic weight reduction and a decrease in spontaneous tumor incidence in mice. With respect to weight reduction we have conducted an extensive gene expression array study evaluating tissue taken from age-matched control mice and mice on Tempol food supplementation for 1 month or 1 year. A number of genes have been identified in Tempol supplemented mice that are differentially up- or down-regulated in liver and brain tissue. Finally, Tempol administration also significantly delays the onset of tumors in Atm and p53 deficient mice and more recently in Fanconis Anemia knockout mice. We have recently found that systemic levels of IGF-1 are decreased in Tempol treated animals, similar to that observed in caloric restricted animals. Gene expression alterations selected tissues from control versus Tempol-treated mice are currently being evaluated. These studies will hopefully enable us to better understand the complex cellular/molecular mechanisms of nitroxides that trigger responses important in the antioxidant properties of nitroxides as well as those related to weight and the chemopreventive findings.